Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6848—Methods of protein analysis involving mass spectrometry
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6848—Methods of protein analysis involving mass spectrometry
  • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J49/00—Particle spectrometers or separator tubes
  • H01J49/02—Details
  • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2458/00—Labels used in chemical analysis of biological material
  • G01N2458/15—Non-radioactive isotope labels, e.g. for detection by mass spectrometry

Definitions

• This invention relates to useful compounds for labelling molecules of interest, particularly biomolecules such as peptides and proteins. Specifically this invention relates to labelling of analytes for detection by mass spectrometry and associated methods of analysing mass labelled analytes by mass spectrometry.
• Narious methods of labelling molecules of interest are known in the art, including radioactive atoms, fluorescent dyes, luminescent reagents, electron capture reagents and light absorbing dyes.
• Each of these labelling systems has features which make it suitable for certain applications and not others.
• interest in non-radioactive labelling systems lead to the widespread commercial development of fluorescent labelling schemes particularly for genetic analysis.
• Fluorescent labelling schemes permit the labelling of a relatively small number of molecules simultaneously, typically 4 labels can be used simultaneously and possibly up to eight.
• the costs of the detection apparatus and the difficulties of analysing the resultant signals limit the number of labels that can be used simultaneously in a fluorescence detection scheme.
• Indirect detection means that an associated label molecule can be used to identify the original analyte, where the label is designed for sensitive detection and a simple mass spectrum.
• Simple mass spectra mean that multiple labels can be used to analyse multiple analytes simultaneously.
• PCT/GB98/00127 describes arrays of nucleic acid probes covalently attached to cleavable labels that are detectable by mass spectrometry which identify the sequence of the covalently linked nucleic acid probe.
• the labelled probes of this application have the structure Nu-L-M where Nu is a nucleic acid covalently linked to L, a cleavable linker, covalently linked to M, a mass label.
• Preferred cleavable linkers in this application cleave within the ion source of the mass spectrometer.
• Preferred mass labels are substituted poly- aryl ethers.
• PCT/GB94/01675 disclose ligands, and specifically nucleic acids, cleavably linked to mass tag molecules. Preferred cleavable linkers are photo-cleavable. These application discloses Matrix Assisted Laser Desorption Ionisation (MALDI) Time of Flight (TOF) mass spectrometry as a specific method of analysing mass labels by mass spectrometry.
• MALDI Matrix Assisted Laser Desorption Ionisation
• TOF Time of Flight
• PCT/US97/22639 discloses releasable non- volatile mass-label molecules.
• these labels comprise polymers, typically biopolymers which are cleavably attached to a reactive group or ligand, i.e. a probe.
• Preferred cleavable linkers appear to be chemically or enzymarically cleavable.
• MALDI TOF mass spectrometry as a specific method of analysing mass labels by mass spectrometry.
• PCT/US97/01070, PCT/US97/01046, and PCT US97/01304 disclose ligands, and specifically nucleic acids, cleavably linked to mass tag molecules. Preferred cleavable linkers appear to be chemically or photo-cleavable. These application discloses a variety of ionisation methods and analysis by quadrupole mass analysers, TOF analysers and magnetic sector instruments as specific methods of analysing mass labels by mass spectrometry. None of these prior art applications mention the use of tandem or serial mass analysis for use in analysing mass labels.
• Two samples can be compared quantitatively by labelling one sample with the biotin linker and labelling the second sample with a deuterated form of the biotin linker.
• Each peptide in the samples is then represented as a pair of peaks in the mass spectrum. Integration of the peaks in the mass spectrum corresponding to each tag indicate the relative expression levels of the peptide linked to the tags.
• This 'isotope encoding' method has a number of limitations.
• a first is the reliance on the presence of thiols in a protein - many proteins do not have thiols while others have several.
• linkers may be designed to react with other side chains, such as amines.
• side chains such as amines.
• many proteins conta i more than one lysine residue multiple peptides per protein would generally be isolated in this approach. It is likely that this would not reduce the complexity of the sample sufficiently for analysis by mass spectrometry.
• a sample that contains too many species is likely to suffer from 'ion suppression', in which certain species ionise preferentially over other species which would normally appear in the mass spectrum in a less complex sample.
• capturing proteins by their side chains is likely to give either too many peptides per protein or certain proteins will be missed altogether.
• the second limitation of this approach is the method used to compare the expression levels of proteins from different samples. Labelling each sample with a different isotope variant of the affinity tag results in an additional peak in the mass spectrum for each peptide in each sample. This means that if two samples are analysed together there will be twice as many peaks in the spectrum. Similarly, if three samples are analysed together, the spectrum will be three times more complex than for one sample alone. It is clear that this approach will be limited, since the ever increasing numbers of peaks will increase the likelihood that two different peptides will have overlapping peaks in the mass spectrum.
• the mass spectra generated for analyte material are very sensitive to contaminants. Essentially, any material introduced into the mass spectrometer that can ionise will appear in the mass spectrum. This means that for many analyses it is necessary to carefully purify the analyte before introducing it into the mass spectrometer. For the purposes of high throughput systems for indirect analysis of analytes through mass labels it would be desirable to avoid any unnecessary sample preparation steps. That is to say it would be desirable to be able to detect labels in a background of contaminating material and be certain that the peak that is detected does in fact correspond to a label.
• the prior art does not disclose methods or compositions that can improve the signal to noise ratio achievable in mass spectrometry based detection systems or that can provide confirmation that a mass peak in a spectrum was caused by the presence of a mass label.
• the invention provides a set of two or more mass labels, each label in the set comprising a mass marker moiety attached via at least one amide bond to a mass normalisation moiety, wherein the aggregate mass of each label in the set may be the same or different and the mass of the mass marker moiety of each label in the set may be the same or different, and wherein in any group of labels within the set having a mass marker moiety of a common mass each label has an aggregate mass different from all other labels in that group, and wherein in any group of labels within the set having a common aggregate mass each label has a mass marker moiety having a mass different from that of all other mass marker moieties in that group, such that all of the mass labels in the set are distinguishable from each other by mass spectrometry, and wherein the mass marker moiety comprises an amino acid and the mass normalisation moiety comprises an amino acid.
• mass marker moiety used in the present context is intended to refer to a moiety that is to be detected by mass spectrometry
• mass normalisation moiety used in the present context is intended to refer to a moiety that is not necessarily to be detected by mass spectrometry, but is present to ensure that a mass label has a desired aggregate mass.
• the number of labels in the set is not especially limited, provided that the set comprises a plurality of labels. However, it is preferred if the set comprises two or more, three or more, four or more, or five or more labels.
• the present invention also provides an array of mass labels, comprising two or more sets of mass labels as defined above, wherein the aggregate mass of each of the mass labels in any one set is different from the aggregate mass of each of the mass labels in every other set in the array.
• the mass marker moiety and the mass normalisation moiety both comprise at least one amino acid.
• the moieties may comprises further groups, if desired, such as more amino acid groups, and/or aryl ether groups.
• the moieties may be modified amino acids, or may be peptides.
• the masses of the different sets in the array may be distinguished by adding further amino acid groups to either or both of the moieties as required.
• a method of analysis comprises detecting an analyte by identifying by mass spectrometry a mass label or a combination of mass labels unique to the analyte, wherein the mass label is a mass label from a set or an array of mass labels as defined above.
• the mass tags may comprise reactive functionalities which facilitate the attachment of the mass tags to analyte molecules.
• the tags in this embodiment are preferably of the following form:
• the array of tags are preferably all chemically identical and the masses of the mass normalisation and mass marker moieties (e.g. amino acid 1 and acid 2 above) are altered by isotope substitutions.
• the tags may comprise a sensitivity enhancing group.
• the tags are preferably of the form:
• the sensitivity enhancing group is usually attached to the mass marker moiety, since it is intended to increase the sensitivity of the detection of this moiety in the mass spectrometer.
• the reactive functionality is shown as being present and attached to a different moiety than the sensitivity enhancing group.
• the tags need not be limited in this way and in some cases comprise the sensitivity enhancing group without the reactive functionality.
• the sensitivity enhancing group may be attached to the same moiety as the reactive functionality.
• the mass tags comprise an affinity capture reagent.
• the affinity capture ligand is biotin.
• the affinity capture ligand allows labelled analytes to be separated from unlabelled analytes by capturing them, e.g. on an avidinated solid phase.
• the invention provides a method of analysing a biomolecule or a mixture of biomolecules.
• This method preferably comprises the steps of: 1. Reacting the biomolecule or mixture of biomolecules with a mass marker according to this invention;
• the affinity tagged biomolecules may be captured by a counter-ligand to allow labelled biomolecules to be separated from unlabelled biomolecules. This step preferably takes place prior to the optional second step above.
• the step of selecting the ions of a predetermined mass to charge ratio is performed in the first mass analyser of a serial instrument.
• the selected ions are then channelled into a separate collision cell where they are collided with a gas or a solid surface according to the fourth step of the first aspect of the invention.
• the collision products are then channelled into a further mass analyser of a serial instrument to detect collision products according to the fifth step of the first aspect of this invention.
• Typical serial instruments include triple quadrupole mass spectrometers, tandem sector instruments and quadrupole time of flight mass spectrometers.
• the step of selecting the ions of a predetermined mass to charge ratio, the step of colliding the selected ions with a gas and the step of detecting the collision products are performed in the same zone of the mass spectrometer. This may effected in ion trap mass analysers and Fourier Transform Ion Cyclotron Resonance mass spectrometers, for example.
• this invention provides sets or arrays of mass labelled molecules of the form:
• the linker is a linker as described below and analyte may be any analyte of interest such as a biomolecule.
• analytes one, more than one or even all the analytes in the set or array are standard analytes with a known mass or with predetermined chromatographic properties. Such standards can be employed in the methods of the present invention for comparison with unknown analytes, for example when analysing the results of a chromatographic separation step.
• This invention describes mass markers that may be readily produced in a peptide synthesiser.
• the compounds used in this invention comprises peptides and modified peptides.
• Peptide synthesis provides chemical diversity allowing for a wide range of markers with chosen properties to be produced in an automated fashion.
• MS/MS' in the context of mass spectrometers refers to mass spectrometers capable of selecting ions, subjecting selected ions to Collision Induced Dissociation (CID) and subjecting the fragment ions to further analysis.
• CID Collision Induced Dissociation
• serial instrument refers to mass spectrometers capable of MS/MS in which mass analysers are organised in series and each step of the MS/MS process is performed one after the other in linked mass analysers.
• Typical serial instruments include triple quadrupole mass spectrometers, tandem sector instruments and quadrupole time of flight mass spectrometers.
• Figure 1 shows a set of 3 mass tags derived from lysine
• Figure 2 shows a set of 5 mass tags derived from alanine
• Figure 3 shows a set of 5 mass tags derived from alanine and tyrosine
• Figure 4 shows a set of 4 mass tags derived from fluorinated forms of phenylglycine
• Figure 5 shows a set of 4 mass tags derived from fluorinated forms of phenylglycine and phenylalanine
• Figure 6a shows a set of 2 affinity ligand mass tags derived from methionine with a hydrazide functionality for labelling carbohydrates
• Figure 6b shows a set of 2 affinity ligand mass tags derived from methionine with a boronic acid functionality for labelling carbohydrates
• Figure 7 shows a set of 2 affinity ligand mass tags derived from methionine with a thiol functionality for labelling dehydroalanine and methyldehydroalanine residues;
• Figure 8 shows a set of 2 affinity ligand mass tags derived from methionine with a maleimide functionality for labelling free thiols
• Figure 9a shows a synthetic pathway for the preparation of an FMOC protected, deuterated methionine residue and figure 9b shows a synthetic pathway for the preparation of a reactive linker that can act as a sensitivity enhancer;
• Figure 10 shows a pair of example peptides derived from different isotopic forms of methionine synthesised to demonstrate the features of this invention
• Figure 11 shows an electrospray mass spectrum of a mixture of the two peptides shown in Figure 10;
• Figure 12 shows an electrospray spectrum of the fragmentation of each of the two peptides shown in Figure 10;
• Figure 13 shows a hypothetical fragmentation mechanism that is likely to account for the spectra shown in Figures 12 and 14;
• Figure 14 shows an electrospray spectrum of the fragmentation of a 70:30 mixture of the two peptides shown in Figure 10;
• Figure 15 shows a graph displaying the expected ratios of peptides A and B (Figure 10) against observed ratios of peptides A and B found in a series of ESI-MS/MS analyses of mixtures of A an B;
• Figures 16a- 16c depict proposed fragmentation mechanisms
• Figures 17a-17d illustrate tags which exploit enhancing cleavage at the cleavable amide bond
• FIGS 18a and 18b show the structures of two versions of the TMT markers
• Figure 19a and 19b show typical CID spectra for a peptide labelled with the first generation TMT at collision energies of 40N (Figure 19a) and 70N ( Figure 19b);
• Figure 20a 20b and 20c show MS and MS/MS spectra for triply charged ions of the peptide 2 (see Table 7) labelled with the first and second generation TMTs;
• Figure 21 shows a typical CID spectra for a peptide (peptide 2 in Table 7) labelled with a second generation TMT;
• Figure 22 shows that the charge state of the TMT tagged peptide does not affect the appearance of the tag fragments in the CID spectra of the labelled peptides;
• Figure 23 shows peptide mixtures with the expected and measured abundance ratios for both the first and second generation tags
• Figure 24 shows the co-elution of each peptide pair, peptides A and B for each peptide from Table 7;
• Figure 25 shows a dynamic range study of TMT peptide pairs 3A/3B, which are present in a ratio of 40:60 and have been analysed at dilutions in the range from 100 frnole to lOOpmole;
• Figures 26a 26b and 26c show the results of a spiking experiment in which peptides pairs 3 A and 3B (500 ftnol in total, in a ratio of 40:60 respectively) bearing a second generation TMT was mixed with a tryptic digest of Bovine Serum Albumin (2 pmol).
• Figures 1 to 5 illustrate a number of important features of the tags of this invention.
• the tags in all of figures 1 to 5 are shown linked to a 'reactive functionality', which could be a linker to an N-hydroxysuccinimide ester for example or any of a number other possibilities some of which are discussed below.
• Figures 1, 2 and 4 show that a number of tags can be generated by combining different mass modified forms of the same amino acid into a series of dipeptides.
• Figures 3 and 5 show sets of tags, which are created by combining different amino acids in heterodimers.
• Figures 1 to 3 illustrate tags, which all have the same total mass and which are chemically identical.
• FIG. 1 shows 3 homodimers of lysine.
• the lysine has been blocked at the epsilon amino groups with methylsulphonyl chloride.
• the sulphonamide linkage is more resistant to fragmentation than a conventional amide linkage, so that the capping group will not be lost when the tag is fragmented in a mass spectrometer using collision induced dissociation at energies sufficient to cleave the conventional backbone amide bond between the pair of modified lysine residues.
• the capping group is used to inhibit protonation at the epsilon position during ionisation of the tags in a mass spectrometer.
• the capped lysine can be prepared prior to synthesis of the mass tags.
• the epsilon amino group can be selectively modified by coupling the amino acid with methylsulphonyl chloride in the presence of copper ions, for example. Airiine and acid functionalities at the alpha position can form chelates with various divalent cations making the alpha amino group unreactive.
• the alpha-amino group of the dipeptide has been converted to a guanidino-group to promote protonation at this position in the tag during ionisation in a mass spectrometer and to differentiate the mass of the fragmentation product from the second alanine residue and natural alanine residues in protein.
• the guanidination of the alpha-position can be performed as the last step of a conventional peptide synthesis before deprotection of the peptide and cleavage from the resin (Z. Tian and R.W. Roeske, Int. J. Peptide Protein Res. 37: 425-429, "Guanidination of a peptide side chain amino group on a solid support", 1991).
• each of the three tags is the same but the N-terminal lysine in each tag differs from the other two by at least four Daltons. This mass difference is usually sufficient to prevent natural isotope peaks from fragmented portions of each tag from overlapping in the mass spectrum with the isotope peaks of the fragmented portions of other tags.
• Figure 2 shows 5 homodimers of alanine. Different isotopically substituted forms of alanine would be used to prepare the five different tags. The total mass of each of the five tags is the same but the N-terminal alanine in each tag differs from the other four by at least one Dalton. The alpha amino group of the dipeptide tag has been methylated to differentiate the fragmentation product of this amino acid from the fragmentation product of the second alanine residue and the natural alanine residues in the protein and to promote protonation at this position in the tag during ionisation in a mass spectrometer.
• Figure 3 shows 5 heterodimers of alanine and tyrosine. Different isotopically substituted forms of alanine and tyrosine would be used to prepare the five different tags.
• the total mass of each of the five tags is the same but the N-terminal alanine in each tag differs from the other four by at least one dalton.
• the alpha amino group of the dipeptide tag has been methylated to differentiate the fragmentation product of this amino acid from the fragmentation products of natural alanine residues in the protein and to promote protonation at this position in the tag during ionisation in a mass spectrometer.
• Figure 4 shows 4 dimers of phenylglycine. Different fluorine substituted forms of phenylglycine would be used to prepare the 4 different tags.
• the total mass of each of the 4 tags is the same but the N-terminal phenylglycine in each tag differs from the other 3 tags by the mass of at least one fluorine atom.
• the alpha amino group of the dipeptide tag has been methylated to differentiate the fragmentation product of this amino acid from the fragmentation product of the second phenylglycine residue and to promote protonation at this position in the tag during ionisation in a mass spectrometer.
• Figure 5 shows 4 dimers comprising phenylglycine and phenylalanine. Different fluorine substituted forms of phenylglycine and phenylalanine would be used to prepare the 4 different tags.
• the total mass of each of the 4 tags is the same but the N-terminal alanine in each tag differs from the other 3 tags by the mass of at least one fluorine atom.
• the alpha amino group of the dipeptide tag has been methylated, although this serves only to protect the amino group from side reactions and to increase protonation as it is not necessary to differentiate the first amino acid as the fragmentation product without methylation would be different from the second amino acid residue of the tag peptide.
• the alpha amino group could be modified to promote protonation at this position in the tag during ionisation in a mass spectrometer by methylation or guanidination if this is desirable.
• the present invention provides a set of mass labels as defined above, in which each label in the set has a mass marker moiety having a common mass and each label in the set has a unique aggregate mass.
• each label in the set has a common aggregate mass and each label in the set has a mass marker moiety of a unique mass.
• the set of labels need not be limited to the two preferred embodiments described above, and may for example comprise labels of both types, provided that all labels are distinguishable by mass spectrometry, as outlined above.
• each mass marker moiety in the set has a common basic structure and each mass normalisation moiety in the set has a common basic structure, and each mass label in the set comprises one or more mass adjuster moieties, the mass adjuster moieties being attached to or situated within the basic structure of the mass marker moiety and/or the basic structure of the mass normalisation moiety.
• every mass marker moiety in the set comprises a different number of mass adjuster moieties and every mass label in the set has the same number of mass adjuster moieties.
• skeleton or backbone may be for example comprise one or more amino acids.
• the skeleton comprises a number of amino acids linked by amide bonds.
• other units such as aryl ether units may also be present.
• the skeleton or backbone may comprise substituents pendent from it, or atomic or isotopic replacements within it, without changing the common basic structure.
• a set of mass labels of the second type referred to above comprises mass labels with the formula:
• M is the mass normalisation moiety
• X is the mass marker moiety
• A is a mass adjuster moiety
• L is the cleavable linker comprising the amide bond
• y and z are integers of 0 or greater
• y+z is an integer of 1 or greater.
• M is a fragmentation resistant group
• L is a linker that is susceptible to fragmentation " on collision with another molecule or atom
• X is preferably a pre-ionised, fragmentation resistant group.
• the sum of the masses of M and X is the same for all members of the set.
• M and X have the same basic structure or core structure, this structure being modified by the mass adjuster moieties.
• the mass adjuster moiety ensures that the sum of the masses of M and X in is the same for all mass labels in a set, but ensures that each X has a distinct (unique) mass.
• the present invention also encompasses arrays of a plurality of sets of mass labels.
• the arrays of mass labels of the present invention are not particularly limited, provided that they contain a plurality of sets of mass labels according to the present invention. It is preferred that the arrays comprise two or more, three or more, four or more, or five or more sets of mass labels.
• each mass label in the array has either of the following structures:
• S is a mass series modifying group
• M is the mass normalisation moiety
• X is the mass marker moiety
• A is the mass adjuster moiety
• L is the cleavable linker comprising the amide bond
• x is an integer of 0 or greater
• y and z are integers of 0 or greater
• y+z is an integer of 1 or greater.
• the mass series modifying group separates the masses of the sets from each other. This group may be any type of group, but is preferably an amino acid, or aryl ether group. Sets may be separated in mass by comprising a different number of amino acids in their moieties than other tags from different sets.
• linker groups which may be used to connect molecules of interest to the mass label compounds of this invention.
• a variety of linkers is known in the art which may be introduced between the mass labels of this invention and their covalently attached analyte. Some of these linkers may be cleavable. Oligo- or poly-ethylene g ⁇ ycols or then derivatives may be used as linkers, such as those disclosed in Maskos, U. & Southern, E.M. Nucleic Acids Research 20: 1679 -1684, 1992.
• Succinic acid based linkers are also widely used, although these are less preferred for applications involving the labelling of oligonucleotides as they are generally base labile and are thus incompatible with the base mediated de-protection steps used in a number of oligonucleotide synthesisers.
• Propargylic alcohol is a bifunctional linker that provides a linkage that is stable under the conditions of oligonucleotide synthesis and is a preferred linker for use with this invention in relation to oligonucleotide applications.
• 6-aminohexanol is a useful bifunctional reagent to link appropriately functionalised molecules and is also a preferred linker.
• cleavable linker groups may be used in conjunction with the compounds of this invention, such as photocleavable linkers.
• Ortho-nitrobenzyl groups are known as photocleavable linkers, particularly 2-nitrobenzyl esters and 2-nitrobenzylamines, which cleave at the benzylamine bond.
• photocleavable linkers see Lloyd- Williams et al, Tetrahedron 49, 11065-11133, 1993, which covers a variety of photocleavable and chemically cleavable linkers.
• WO 00/02895 discloses the vinyl sulphone compounds as cleavable linkers, which are also applicable for use with this invention, particularly in applications involving the labelling of polypeptides, peptides and amino acids. The content of this application is incorporated by reference.
• WO 00/02895 discloses the use of silicon compounds as linkers that are cleavable by base in the gas phase. These linkers are also applicable for use with this invention, particularly in applications involving the labelling of oligonucleotides. The content of this application is incorporated by reference.
• the mass labels of the present invention may comprise reactive functionalities, Re, to help attach them to analytes.
• Re is a reactive functionality or group which allows the mass label to be reacted covalently to an appropriate functional group in an analyte molecule, such as, but not limited to, a nucleotide oligonucleotide, polynucleotide, amino acid, peptide or polypeptide.
• Re may be attached to the mass labels via a linker which may or may not be cleavable.
• a variety of reactive functionalities may be introduced into the mass labels of this invention.
• Table 1 below lists some reactive functionalities that may be reacted with nucleophilic functionalities which are found in biomolecules to generate a covalent linkage between the two entities.
• primary amines or thiols are often introduced at the termini of the molecules to permit labelling. Any of the functionalities listed below could be introduced into the compounds of this invention to permit the mass markers to be attached to a molecule of interest.
• a reactive functionality can be used to introduce a further linker groups with a further reactive functionality if that is desired.
• Table 1 is not intended to be exhaustive and the present invention is not limited to the use of only the listed functionalities. Table 1
• charge carrying functionalities and solubilising groups. These groups may be introduced into the mass labels such as in the mass markers of the invention to promote ionisation and solubility.
• the choice of markers is dependent on whether positive or negative ion detection is to be used.
• Table 2 below lists some functionalities that may be introduced into mass markers to promote either positive or negative ionisation. The table is not intended as an exhaustive list, and the present invention is not limited to the use of only the listed functionalities.
• WO 00/02893 discloses the use of metal-ion binding moieties such as crown-ethers or porphyrins for the purpose of improving the ionisation of mass markers. These moieties are also be applicable for use with the mass markers of this invention.
• the components of the mass markers of this invention are preferably fragmentation resistant so that the site of fragmentation of the markers can be controlled by the introduction of a linkage that is easily broken by Collision Induced Dissociation (CID).
• CID Collision Induced Dissociation
• Aryl ethers are an example of a class of fragmentation resistant compounds that may be used in this invention. These compounds are also chemically inert and thermally stable.
• WO 99/32501 discusses the use of poly-ethers in mass spectrometry in greater detail and the content of this application is incorporated by reference.
• the present invention also provides a set of two or more probes, each probe in the set being different and being attached to a unique mass label or a unique combination of mass labels, from a set or an array of mass labels as defined as defined above.
• an array of probes comprising two or more sets of probes, wherein each probe in any one set is attached to a unique mass label, or a unique combination of mass labels, from a set of mass labels as defined above, and wherein the probes in any one set are attached to mass labels from the same set of mass labels, and each set of probes is attached to mass labels from unique sets of mass labels from an array of mass labels as defined above.
• each probe is preferably attached to a unique combination of mass labels, each combination being distinguished by the presence or absence of each mass label in the set of mass labels and/or the quantity of each mass label attached to the probe. This is termed the "mixing mode" of the present invention, since the probes may be attached to a mixture of mass labels.
• each probe comprises a biomolecule. Any biomolecule can be employed, but the biomolecule is preferably selected from a DNA, an RNA, an oligonucleotide, a nucleic acid base, a peptide, a polypeptide, a protein and an amino acid.
• this invention provides sets and arrays of mass labelled analytes, such as nucleotides, oligonucleotides and polynucleotides, of the form:
• linker is a linker as defined above, and label is a mass label from any of the sets and arrays defined above.
• each analyte comprises a biomolecule.
• Any biomolecule can be employed, but the biomolecule is preferably selected from a DNA, an RNA, an oligonucleotide, a nucleic acid base, a peptide, a polypeptide, a protein and an amino acid.
• each analyte is preferably attached to a unique combination of mass labels, each combination being distinguished by the presence or absence of each mass label in the set of mass labels and/or the quantity of each mass label attached to the probe.
• this is termed the "mixing mode" of the present invention, since the probes may be attached to a mixture of mass labels.
• the present invention provides a method of analysis, which method comprises detecting an analyte by identifying by mass spectrometry a mass label or a combination of mass labels unique to the analyte, wherein the mass label is a mass label from a set or an array of mass labels as defined above.
• the type of method is not particularly limited, provided that the method benefits from the use of the mass labels of the present invention to identify an analyte.
• the method may be, for example, a method of sequencing nucleic acid or a method of profiling the expression of one or more genes by detecting quantities of protein in a sample. The method is especially advantageous, since it can be used to readily analyse a plurality of analytes simultaneously.
• the method also has advantages for analysing single analytes individually, since using the present mass labels, mass spectra which are cleaner than conventional spectra are produced, making the method accurate and sensitive.
• the present invention provides a method which method comprises:
• the mass label is cleaved from the probe prior to detecting the mass label by mass spectrometry.
• the method comprises contacting one or more nucleic acids with a set of hybridisation probes.
• the set of hybridisation probes typically comprises a set of up to 256 4-mers, each probe in the set having a different combination of nucleic acid bases. This method may be suitable for identifying the presence of target nucleic acids, or alternatively can be used in a stepwise method of primer extension sequencing of one or more nucleic acid templates.
• the mass labels of the present invention are particularly suitable for use in methods of 2-dimensional analysis, primarily due to the large number of labels that can be simultaneously distinguished.
• the labels may thus be used in a method of 2-dimensional gel electrophoresis, or in a method of 2-dimensional mass spectrometry.
• peptide mass tags of this invention will be possible using conventional peptide synthesis methods and commercially available reagents. Modified amino acids that are not commercially available are also contemplated for the synthesis of further peptide mass tags.
• Modern peptide synthesis is typically carried out on solid phase supports in automated synthesiser instruments, which deliver all the necessary reagents for each step of a peptide synthesis to the solid support and remove spent reagents and unreacted excess reagents at the end of each step in the cycle.
• Solid phase peptide synthesis is, however, often performed manually, particularly when specialist reagents are being tested for the first time. In essence peptide synthesis involves the addition of N-protected amino acids to the solid support.
• the peptide is normally synthesised with the C-terminal carboxyl group of the peptide attached to the support, and the sequence of the peptide is built from the C- terminal arnii o acid to the N-terminal amino acid.
• the C-terminal amino acid is coupled to the support by a cleavable linkage.
• the N-protected alpha amino group of each amino acid is deprotected to allow coupling of the carboxyl group of the next amino acid to the growing peptide on the solid support.
• peptide synthesis is performed by one of two different synthetic procedures, which are distinguished by the conditions needed to remove the N-protecting group.
• t-BOC tert-butyloxycarbonyl
• FMOC fluorenylmethoxycarbonyl
• Reactive side chains in amino acids also need protection during cycles of amide bond formation.
• side chains include the epsilon amino group of lysine, the guanidino side-chain of arginine, the thiol functionality of cysteine, the hydroxyl functionalities of serine, threonine and tyrosine, the indole ring of tryptophan and the imidazole ring of histidine.
• the choice of protective groups used for side-chain protection is determined by the cleavage conditions of the alpha-amino protection groups, as the side-chain protection groups must be resistant to the deprotection conditions used to remove the alpha-amino protection groups.
• a first protective group is said to be 'orthogonal' to a second protective group if the first protective group is resistant to deprotection under the conditions used for the deprotection of the second protective group and if the deprotection conditions of the first protecting group do not cause deprotection of the second protecting group.
• a variety of amino acids can be used in the mass marker moiety and the mass normalisation moiety. Neutral amino acids are preferred in the mass normalisation moiety and charged amino acids are preferred in the mass marker moieties (since this facilitates ionisation and increases sensitivity) e.g. in the position marked amino acid 1 and amino acid 2 in the first and fourth embodiments of this invention.
• a number of commercially available isotopically mass modified amino acids are shown in Table 5 below. Any combination of 1, 2 ,3, or 4 or more amino acids from this list are preferred in each of the moieties according to the present invention. Table 5
• both the D- and L- forms are available (from ISOTEC Inc., Miamisburg, Ohio for example), either of which may be used in the preparation of the tags of this invention. Mixtures of D and L forms are also available but are less preferred if the tags of this invention are to be used in chromatographic separations. For some, FMOC or t-BOC protected derivatives are also available. Mass modified amino acids based on substitution of deuterium for hydrogen and on substitution of 13 C and 15 N isotopes for 12 C and 13 N isotopes are also available and are equally applicable for the synthesis of the tags of this invention.
• Narious amino acids that are not typically found in peptides may also be used in the tags of this invention, for example deuterated forms of amino-butyric acid are commercially available.
• non-radioactive, stable isotopes are preferred for safety reasons but there is no necessary limitation to stable isotopes.
• Fluorinated derivatives of a number of amino acids are also available. Some of the commercially available fluorinated amino acids are shown in Table 6 below.
• the reagents are available as mixtures of D and L forms.
• fluorinated variants of amino acids are less preferred than isotope substituted variants.
• the fluorinated compounds can be used to generate a range of mass tags with the same mass but each tag will be chemically different, which means that their behaviour in the mass spectrometer will vary more than isotope substituted tags.
• the tags will not have identical chromatographic properties if the tags are to be used in chromatographic separations.
• the mass tags of the invention comprise a reactive functionality, hi the simplest embodiments this may be an N- hydroxysuccinimide ester introduced by activation of the C-terminus of the tag peptides of this invention. In conventional peptide synthesis, this activation step would have to take place after the peptide mass tag has been cleaved from the solid support used for its synthesis.
• An N-hydroxysuccinimide activated peptide mass tag could also be reacted with hydrazine to give a hydrazide reactive functionality, which can be used to label periodate oxidised sugar moieties, for example.
• Amino-groups or thiols can be used as reactive functionalities in some applications and these may be introduced by adding lysine or cysteine after amino acid 2 of the tag peptide.
• Lysine can be used to couple tags to free carboxyl functionalities using a carbodiimide as a coupling reagent. Lysine can also be used as the starting point for the introduction of other reactive functionalities into the tag peptides of this invention.
• the thiol-reactive maleimide functionality can be introduced by reaction of the lysine epsilon amino group with maleic anhydride.
• the cysteine thiol group can be used as the starting point for the synthesis of a variety of alkenyl sulphone compounds, which are useful protein labelling reagents that react with thiols and amines.
• Compounds such as aminohexanoic acid can be used to provide a spacer between the mass modified amino acids and the reactive functionality.
• the mass markers comprise an affinity capture ligand.
• Affinity capture ligands are ligands, which have highly specific binding partners. These binding partners allow molecules tagged with the ligand to be selectively captured by the binding partner.
• a solid support is derivitised with the binding partner so that affinity ligand tagged molecules can be selectively captured onto the solid phase support.
• a preferred affinity capture ligand is biotin, which can be introduced into the peptide mass tags of this invention by standard methods known in the art. In particular a lysine residue may be incorporated after amino acid 2 through which an amine-reactive biotin can be linked to the peptide mass tags ( see for example Geahlen R.L.
• affinity capture ligands include digoxigenin, fluorescein, nitrophenyl moieties and a number of peptide epitopes, such as the c-myc epitope, for which selective monoclonal antibodies exist as counter-ligands.
• Metal ion binding ligands such as hexahistidine, which readily binds Ni 2+ ions, are also applicable.
• Chromatographic resins which present iminodiacetic acid chelated Ni 2+ ions are commercially available, for example. These immobilised nickel columns may be used to capture peptide mass tags, which comprise oligomeric histidine.
• an affinity capture functionality may be selectively reactive with an appropriately derivitised solid phase support.
• Boronic acid for example, is known to selectively react with vicinal cis-diols and chemically similar ligands, such as salicylhydroxamic acid.
• Reagents comprising boronic acid have been developed for protein capture onto solid supports derivitised with salicylhydroxamic acid (Stolowitz MX., et al., Bioconjug Chem 12(2): 229-239, "Phenylboronic Acid- Salicylhydroxamic Acid Bioconjugates. 1. A Novel Boronic Acid Complex for Protein Immobilization.” 2001; Wiley J.P.
• the use of this sort of chemistry would not be directly compatible with biomolecules bearing vicinal cis-diol- containing sugars, however these sorts of sugars could be blocked with phenylboronic acid or related reagents prior to reaction with boronic acid derivitised peptide mass tag reagents.
• Mass Spec Sensitivity Enhancing Groups and Mass Differentiation hi preferred embodiments of the first and fourth aspects of this invention the peptide mass tags comprise Sensitivity Enhancing Groups.
• Figures 1 to 5 illustrate the use of methylation and guanidination as methods of improving sensitivity.
• these Sensitivity Enhancing Groups can differentiate the fragmentation products of the N- terminal amino acid from the fragmentation products of the second amino acid in the peptide tag and natural amino acid residues in the protein, if this is the same as the first amino acid.
• the sensitivity enhancing group can also distinguish the fragmentation products of the N-terminal amino acid of the peptide mass tag from the fragmentation products of natural amino acids when the tags of this invention are used to label peptides and proteins.
• the guanidino group and the tertiary amino group are both useful Sensitivity Enhancing Groups for electrospray mass spectrometry.
• Each type of Sensitivity Enhancing Group has different benefits, which depend on the method of ionisation used and on the methods of mass analysis used.
• the mechanism by which sensitivity is enhanced may also be different for each type of group.
• Some derivitisation methods increase basicity and thus promote protonation and charge localisation, while other methods increase surface activity of the tagged peptides, which improves sensitivity in surface desorption techniques like Matrix Assisted Laser Desorption Ionisation (MALDi) and Fast Atom Bombardment (FAB).
• MALDi Matrix Assisted Laser Desorption Ionisation
• FAB Fast Atom Bombardment
• Negative ion mass spectrometry is often more sensitive because there is less background noise.
• Charge derivitisation can also change the fragmentation products of derivatised peptides, when collision induced dissociation is used.
• a chromatographic or electrophoretic separation is preferred to reduce the complexity of the sample prior to analysis by mass spectrometry.
• mass spectrometry techniques are compatible with separation technologies particularly capillary zone elecfrophoresis and High Performance Liquid Chromatography (HPLC).
• HPLC High Performance Liquid Chromatography
• the choice of ionisation source is limited to some extent if a separation is required as ionisation techniques such as MALDI and FAB (discussed below) which ablate material from a solid surface are less suited to chromatographic separations.
• MALDI and FAB ablate material from a solid surface are less suited to chromatographic separations.
• Dynamic FAB and ionisation techniques based on spraying such as electrospray, thermospray and APCI are all readily compatible with in-line chromatographic separations and equipment to perform such liquid chromatography mass spectrometry analysis is commercially available.
• ESI-MS Electrospray Ionisation Mass Spectrometry
• FAB Fast Atom Bombardment
• MALDI MS Matrix Assisted Laser Desorption Ionisation Mass Spectrometry
• APCI-MS Atmospheric Pressure Chemical Ionisation Mass Spectrometry
• Electrospray ionisation requires that the dilute solution of the analyte molecule is 'atomised' into the spectrometer, i.e. injected as a fine spray.
• the solution is, for example, sprayed from the tip of a charged needle in a stream of dry nitrogen and an electrostatic field.
• the mechanism of ionisation is not fully understood but is thought to work broadly as follows, hi a stream of nitrogen the solvent is evaporated. With a small droplet, this results in concentration of the analyte molecule. Given that most biomolecules have a net charge this increases the electrostatic repulsion of the dissolved molecule. As evaporation continues this repulsion ultimately becomes greater than the surface tension of the droplet and the droplet disintegrates into smaller droplets.
• This process is sometimes referred to as a 'Coulombic explosion'.
• the electrostatic field helps to further overcome the surface tension of the droplets and assists in the spraying process.
• the evaporation continues from the smaller droplets which, in turn, explode iteratively until essentially the biomolecules are in the vapour phase, as is all the solvent.
• This technique is of particular importance in the use of mass labels in that the technique imparts a relatively small amount of energy to ions in the ionisation process and the energy distribution within a population tends to fall in a narrower range when compared with other techniques.
• the ions are accelerated out of the ionisation chamber by the use of electric fields that are set up by appropriately positioned electrodes.
• the polarity of the fields may be altered to extract either negative or positive ions.
• the potential difference between these electrodes determines whether positive or negative ions pass into the mass analyser and also the kinetic energy with which these ions enter the mass spectrometer. This is of significance when considering fragmentation of ions in the mass specfrometer. The more energy imparted to a population of ions me more likely it is that fragmentation will occur through collision of analyte molecules with the bath gas present in the source.
• By adjusting the electric field used to accelerate ions from the ionisation chamber it is possible to control the fragmentation of ions. This is advantageous when fragmentation of ions is to be used as a means of removing tags from a labelled biomolecule.
• Electrospray ionisation is particularly advantageous as it can be used in-line with liquid chromatography, referred to as Liquid Chromatography Mass Spectrometry (LC-MS).
• MALDI Matrix Assisted Laser Desorption Ionisation
• MALDI requires that the biomolecule solution be embedded in a large molar excess of a photo-excitable 'matrix'.
• the application of laser light of the appropriate frequency results in the excitation of the mafrix which in turn leads to rapid evaporation of the matrix along with its entrapped biomolecule.
• Proton transfer from the acidic matrix to the biomolecule gives rise to protonated forms of the biomolecule which can be detected by positive ion mass spectrometry, particularly by Time-Of-Flight (TOF) mass spectrometry.
• TOF Time-Of-Flight
• Negative ion mass spectrometry is also possible by MALDI TOF. This technique imparts a significant quantity of franslational energy to ions, but tends not to induce excessive fragmentation despite this. Accelerating voltages can again be used to control fragmentation with this technique though.
• FAB Fast Atom Bombardment
• FAB techniques are also compatible with liquid phase inlet systems - the liquid eluting from a capillary elecfrophoresis inlet or a high pressure liquid chromatography system pass through a frit, essentially coating the surface of the frit with analyte solution which can be ionised from the frit surface by atom bombardment.
• Fragmentation of peptides by collision induced dissociation is used in this invention to identify tags on proteins.
• Narious mass analyser geometries may be used to fragment peptides and to determine the mass of the fragments.
• Tandem mass spectrometers allow ions with a pre-determined mass-to-charge ratio to be selected and fragmented by collision induced dissociation (CID). The fragments can then be detected providing structural information about the selected ion.
• CID collision induced dissociation
• characteristic cleavage patterns are observed, which allow the sequence of the peptide to be determined.
• Natural peptides typically fragment randomly at the amide bonds of the peptide backbone to give series of ions that are characteristic of the peptide.
• CID fragment series are denoted a n , b n , c n , etc.
• fragment series are denoted x n , y n , z ⁇ , etc. where the charge is retained on the C-terminal fragment of the ion.
• Trypsin and thrombin are favoured cleavage agents for tandem mass spectrometry as they produce peptides with basic groups at both ends of the molecule, i.e. the alpha-amino group at the N-terminus and lysine or arginine side-chains at the C-terminus.
• These doubly charged ions produce both C-terminal and N-terminal ion series after CID. This assists in determining the sequence of the peptide. Generally speaking only one or two of the possible ion series are observed in the CID spectra of a given peptide.
• the b- series of N-terminal fragments or the y-series of C-terminal fragments predominate. If doubly charged ions are analysed then both series are often detected. In general, the y- series ions predominate over the b-series.
• This mechanism requires a carbonyl group from an amide bond adjacent to a protonated amide on the N-temnnal side of the protonated amide to carry out the nucleophilic attack.
• a charged oxazolonium ion gives rise to b-series ions, while proton transfer from the N-terminal fragment to the C- terminal fragment gives rise to y-series ions as shown in figure 16a.
• This requirement for an appropriately located carbonyl group does not account for cleavage at amide bonds adjacent to the N-terminal amino acid, when the N-te ⁇ ninus is not protected and, in general, b-series ions are not seen for the amide between the N-terrninal and second amino acid in a peptide.
• peptides with acetylated N-termfni do meet the structural requirements of this mechanism and fragmentation can take place at the amide bond immediately after the first amino acid by this mechanism.
• Peptides with thioacetylated N-termini will cleave particularly easily by the oxazolone mechanism as the sulphur atom is more nucleophilic than an oxygen atom in the same position. Fragmentation of the amide backbone of a peptide can also be modulated by methylation of the backbone.
• Methylation of an amide nifrogen in a peptide can promote fragmentation of the next amide bond C-terminal to the methylated amide and also favours the formation of b-ions.
• the enhanced fragmentation may be partly due to the electron donating effect of the methyl group increasing the nucleophilicity of the carbonyl group of the methylated amide, while the enhanced formation of b-ions may be a result of the inability of the oxazolonium ion that forms to transfer protons to the C-terminal fragment as shown in figure 16b.
• thioacetylation of the N-terminus of a tag dipeptide can be used to enhance cleavage of the tag peptide at the next amide bond.
• FIGS. 17a and 17b illustrate pairs of tags that exploit these methods of enhancing cleavage at the marked amide linkage.
• proline, and asp-pro linkages can also be used the tag peptides of this invention to promote fragmentation at specified locations within a peptide.
• Figures 17c and 17d illustrate pairs of tags that exploit these methods of enhancing cleavage at the marked amide linkage.
• Figure 17c illustrates a pair of fripeptide tags with the sequence aianine-proline-alanine.
• the proline linkage promotes cleavage at its ⁇ -terminal amide. This is enhanced by the presence of a thioacetyl protecting group at the ⁇ -terminus of the fripeptide and the cleavability is further enhanced by methylation of the ⁇ -terminal nitrogen.
• the tags have the same mass but in the first tag there is an alanine residue with heavy isotopes in the third position of the fripeptide while in the second tag there is an alanine residue with heavy isotopes in the first position of the fripeptide.
• Figure 17d illustrates a pair of fripeptide tags with the sequence aspartic acid-proline-alanine. The proline linkage promotes cleavage at its ⁇ - terminal amide. This is enhanced by the presence of the aspartic acid residue. The ⁇ - terminus of the fripeptide is methylated to promote localised protonation here.
• the tags have the same mass but in the first tag there is an alanine residue with heavy isotopes in the third position of the fripeptide while in the second tag there is an aspartic acid residue with heavy isotopes in the first position of the fripeptide.
• a typical tandem mass spectrometer geometry is a triple quadrupole which comprises two quadrupole mass analysers separated by a collision chamber, also a quadrupole.
• This collision quadrupole acts as an ion guide between the two mass analyser quadrupoles.
• a gas can be introduced into the collision quadrupole to allow collision with the ion sfream from the first mass analyser.
• the first mass analyser selects ions on the basis of their mass/charge ration which pass through the collision cell where they fragment.
• the fragment ions are separated and detected in the third quadrupole. Induced cleavage can be performed in geometries other than tandem analysers.
• Ion trap mass spectrometers can promote fragmentation through infroduction of a gas into the frap itself with which trapped ions will collide.
• Ion traps generally contain a bath gas, such as helium but addition of neon for example, promotes fragmentation. Similarly photon induced fragmentation could be applied to trapped ions.
• Another favourable geometry is a Quadrupole/Orthogonal Time of Flight tandem instrument where the high scanning rate of a quadrupole is coupled to the greater sensitivity of a reflectron TOF mass analyser to identify the products of fragmentation.
• a sector mass analyser comprises two separate 'sectors', an electric sector which focuses an ion beam leaving a source into a stream of ions with the same kinetic energy using electric fields.
• the magnetic sector separates the ions on the basis of their mass to generate a spectrum at a detector.
• tandem mass specfrometry a two sector mass analyser of this kind can be used where the electric sector provide the first mass analyser stage, the magnetic sector provides the second mass analyser, with a collision cell placed between the two sectors.
• Two complete sector mass analysers separated by a collision cell can also be used for analysis of mass tagged peptides.
• Ion Trap mass analysers are related to the quadrupole mass analysers.
• the ion frap generally has a 3 electrode construction - a cylindrical electrode with 'cap' electrodes at each end forming a cavity.
• a sinusoidal radio frequency potential is applied to the cylindrical electrode while the cap electrodes are biased with DC or AC potentials.
• Ions injected into the cavity are constrained to a stable circular trajectory by the oscillating electric field of the cylindrical electrode. However, for a given amplitude of the oscillating potential, certain ions will have an unstable trajectory and will be ejected from the frap.
• a sample of ions injected into the trap can be sequentially ejected from the trap according to their mass/charge ratio by altering the oscillating radio frequency potential.
• Ion traps are generally operated with a small quantity of a 'bath gas', such as helium, present n the ion trap cavity. This increases both the resolution and the sensitivity of the device as the ions entering the trap are essentially cooled to the ambient temperature of the bath gas through collision with the bath gas. Collisions both increase ionisation when a sample is introduced into the trap and dampen the amplitude and velocity of ion trajectories keeping them nearer the centre of the trap. This means that when the oscillating potential is changed, ions whose trajectories become unstable gain energy more rapidly, relative to the damped circulating ions and exit the trap in a tighter bunch giving a narrower larger peaks..
• a 'bath gas' such as helium
• Ion traps can mimic tandem mass spectrometer geometries, in fact they can mimic multiple mass spectrometer geometries allowing complex analyses of trapped ions.
• a single mass species from a sample can be retained in a trap, i.e. all other species can be ejected and then the retained species can be carefully excited by super-imposing a second oscillating frequency on the first.
• the excited ions will then collide with the bath gas and will fragment if sufficiently excited.
• the fragments can then be analysed further. It is possible to retain a fragment ion for further analysis by ejecting other ions and then exciting the fragment ion to fragment. This process can be repeated for as long as sufficient sample exists to permit further analysis.
• FTICR mass specfrometry has similar features to ion traps in that a sample of ions is retained within a cavity but in FTICR MS the ions are frapped in a high vacuum chamber by crossed electric and magnetic fields.
• the electric field is generated by a pair of plate electrodes that form two sides of a box.
• the box is contained in the field of a superconducting magnet which in conjunction with the two plates, the trapping plates, constrain injected ions to a circular trajectory between the trapping plates, perpendicular to the applied magnetic field.
• the ions are excited to larger orbits by applying a radio- frequency pulse to two 'transmitter plates' which form two further opposing sides of the box.
• the cycloidal motion of the ions generate corresponding electric fields in the remaining two opposing sides of the box which comprise the 'receiver plates'.
• the excitation pulses excite ions to larger orbits which decay as the coherent motions of the ions is lost through collisions.
• the corresponding signals detected by the receiver plates are converted to a mass spectrum by Fourier Transform (FT) analysis.
• FT Fourier Transform
• these instruments can perform in a similar manner to an ion frap - all ions except a single species of interest can be ejected from the trap.
• a collision gas can be introduced into the trap and fragmentation can be induced.
• the fragment ions can be subsequently analysed.
• fragmentation products and bath gas combine to give poor resolution if analysed by FT analysis of signals detected by the 'receiver plates', however the fragment ions can be ejected from the cavity and analysed in a tandem configuration with a quadrupole, for example.
• labelled biomolecules are subjected to a chromatographic separation prior to analysis by mass specfrometry.
• This is preferably High Performance Liquid Chromatography (HPLC) which can be coupled directly to a mass spectrometer for in-line analysis of the peptides as they elute from the chromatographic column.
• HPLC High Performance Liquid Chromatography
• a variety of separation techniques may be performed by HPLC but reverse phase chromatography is a popular method for the separation of peptides prior to mass spectrometry.
• Capillary zone elecfrophoresis is another separation method that may be coupled directly to a mass spectrometer for automatic analysis of eluting samples.
• the tags are used for the analysis of mixtures of peptides by liquid chromatography tandem mass spectrometry (LC-MS-MS).
• LC-MS-MS liquid chromatography tandem mass spectrometry
• the labelled peptides After attachment of the tags, the labelled peptides will have a mass that is shifted by the mass of the tag.
• the mass of the peptide may be sufficient to identify the source protein.
• the tag needs to be detected which can be achieved by selected reaction monitoring with a triple quadrupole, discussed in more detail below.
• the first quadrupole of the friple quadrupole is set to let through ions whose mass-to-charge ratio corresponds to that of the peptide of interest, adjusted for the mass of the marker.
• the selected ions are then subjected to collision induced dissociation (CID) in the second quadrupole. Under the sort of conditions used in the analysis of peptides the ions will fragment mostly at the amide bonds in the molecule.
• CID collision induced dissociation
• the markers in figures 1 and 2 have an amide bond, which releases the N-terminal portion of the tag on cleavage. Although the tags all have the same mass, the terminal portion is different because of differences in the substituents on either side of the amide bond. Thus the markers can be distinguished from each other.
• the presence of the marker fragment associated with an ion of a specific mass should confirm that the ion was a peptide and the relative peak heights of the tags from different samples will give information about the relative quantities of the peptides in their samples. If the mass is not sufficient to identify a peptide, either because a number of terminal peptides in the sample have the same terminal mass or because the peptide is not known, then sequence information may be determined by analysis of the complete CID spectrum.
• the peptide fragmentation peaks can be used to identify the peptides while the mass tag peaks give information about the relative quantities of the peptides.
• the analysis of proteins by tandem mass spectrometry, particularly mixtures of peptides, is complicated by the 'noisiness' of the spectra obtained.
• Peptides isolated from biological samples are often contaminated with buffering reagents, denaturants and detergents, all of which introduce peaks into the mass spectrum. As a result, there are often more contamination peaks in the spectrum than peptide peaks and identifying peaks that correspond to peptides is major problem, especially with small samples of proteins that are difficult to isolate. As a result various methods are used to determine which peaks correspond to peptides before detailed CID analysis is performed.
• Triple quadrupole based instruments permit 'precursor ion scanning' (see Wilm M. et al., Anal Chem 68(3):527-33, "Parent ion scans of unseparated peptide mixtures.” (1996)).
• the triple quadrupole is operated in 'single reaction monitoring' mode, in which the first quadrupole scans over the full mass range and each gated ion is subjected to CID in the second quadrupole.
• the third quadrupole is set to detect only one specific fragment ion, which is usually a characteristic fragment ion from a peptide such as immonium ions. The presence of phosphate groups can also be detected using this sort of technique.
• An alternative method used with quadrupole/time-of-flight mass spectrometers scans for doubly charged ions by identifying ions which when subjected to CID produce daughter ions with higher mass-to-charge ratios than the parent ion.
• a further method of identifying doubly charged ions is to look for sets of peaks in the spectrum which are only 0.5 daltons apart with appropriate intensity ratios which would indicate that the ions are the same differing only by the proportion of 13 C present in the molecule.
• a novel form of precursor ion scanning may be envisaged in which peptide peaks are identified by the presence of fragments corresponding to the mass labels of this invention after subjecting the labelled peptides to CID.
• the peptides isolated from each sample by the methods of this invention may be labelled with more than one tag.
• An equimolar mixture of a 'precursor ion scanning' tag which is used in all samples and a sample specific tag may be used to label the peptides in each sample. In this way changes in the level of peptides in different samples will not have an adverse effect on the identification of peptide peaks in a precursor ion scan.
• each peptide in the sample there should be a double peak of equal intensity for each peptide where the double peak is 2 Daltons apart. This is complicated slightly by intrinsic peptide isotope peaks but allows for automated scanning of the CID spectrum for doublets. The differences in mass between doublets can be determined to identify the amino acid by the two fragments differ. This method may be applicable with the methods of this invention if N-terminal peptides are isolated.
• mRNA messenger RNA
• peptides may be analysed effectively using the methods of this invention.
• the tags of this invention allow the same peptide from different samples to be identified using LC-MS-MS.
• the relative quantities of the same peptide in different samples may be determined.
• the ability to rapidly and sensitively determine the identity and relative quantities of peptides in a number of samples allows for expression profiling. Therefore it is an object of this invention to provide improved methods for comparative analysis of complex protein samples based on the selective isolation and labelling of peptides.
• Isolation of N- or C-terminal peptides has been described as a method to determine a global expression profile of a protein sample. Isolation of terminal peptides ensures that at least one and only one peptide per protein is isolated thus ensuring that the complexity of the sample that is analysed does not have more components than the original sample. Reducing large polypeptides to shorter peptides makes the sample more amenable to analysis by mass spectrometry. Methods for isolating peptides from the termini of polypeptides are discussed in PCT/GB98/00201, PCT/GB99/03258.
• LC-MS/MS liquid chromatography tandem mass spectrometry
• Two protein samples can be compared by labelling the cysteine residues with a different isotopically modified biotin tag. This approach is slightly more redundant than an approach based on isolating terminal peptides as, on average, more than one peptide per protein is isolated so there are more peptide species in the sample than protein species. This increase in complexity is made worse by the nature of the tags used by Gygi et al.
• FIG. 8 An improved method for analysing protein samples by labelling cysteine residues is envisaged using tags of the form shown in Figure 8.
• This Figure illustrates a pair of improved affinity tags derived from methionine. Different isotopically substituted forms of methionine would be used to prepare the two different tags. The total mass of each of the two tags is the same but the N-terminal methionine in each tag differs from the other tag by three Daltons.
• the alpha amino group of the dipeptide tag has been guanidinated to differentiate the fragmentation product of this amino acid from the fragmentation product of the second methionine residue and the natural methionine residues in protein and to promote protonation at this position in the tag during ionisation in a mass spectrometer, hi addition these tags comprise a thiol reactive maleimide functionality.
• a protocol for the analysis of a protein sample containing polypeptides with cysteine residues comprises the steps of: 1. Reducing and reacting all cysteine residues in at least one protein sample with a maleimide affinity ligand mass tag;
• the protein samples may be digested with the sequence specific endoprotease before or after reaction of the sample with the affinity ligand mass tag.
• a protocol for the analysis of a sample of proteins, which contains carbohydrate modified proteins comprises the steps of:
• a protocol for the analysis of a protein sample containing carbohydrate modified polypeptides comprises the steps of:
• the sample may be digested with the sequence specific endoprotease before or after reaction of the sample with the affinity ligand mass tag.
• Nicinal-diols in sialic acids for example, can also be converted into carbonyl groups by oxidative cleavage with periodate. Enzymatic oxidation of sugars containing te ⁇ riinal galactose or galactosamine with galactose oxidase can also convert hydroxyl groups in these sugars to carbonyl groups. Complex carbohydrates can also be treated with carbohydrate cleavage enzymes, such as neuramidase, which selectively remove specific sugar modifications leaving behind sugars, which can be oxidised. These carbonyl groups can be tagged allowing proteins bearing such modifications to be detected or isolated.
• Hydrazide reagents such as Biocytin hydrazide (Pierce & Warriner Ltd, Chester, UK) will react with carbonyl groups in carbonyl-containing carbohydrate species (E.A. Bayer et al. , Anal. Biochem. 170: 271 - 281, "Biocytin hydrazide - a selective label for sialic acids, galactose, and other sugars in glycoconjugates using avidin biotin technology", 1988).
• a carbonyl group can be tagged with an amine modified biotin, such as Biocytin and EZ-LinkTM PEO-Biotin (Pierce & Warriner Ltd, Chester, UK), using reductive alkylation (Means G.E., Methods Enzymol 47: 469-478, "Reductive alkylation of amino groups.” 1977; Rayment I., Methods Enzymol 276: 171-179, "Reductive alkylation of lysine residues to alter crystallization properties of proteins.” 1997). Proteins bearing vicinal-diol containing carbohydrate modifications in a complex mixture can thus be biotinylated. Biotinylated, hence carbohydrate modified, proteins may then be isolated using an avidinated solid support.
• an amine modified biotin such as Biocytin and EZ-LinkTM PEO-Biotin (Pierce & Warriner Ltd, Chester, UK)
• a set of peptide mass tags according to this invention can be synthesised for the analysis of carbohydrate modified peptides that have been oxidised with periodate, as shown in Figure 6a.
• Figure 6a shows a set of two tags derived from methionine. Different isotopically substituted forms of methionine would be used to prepare the two different tags. The total mass of each of the two tags is the same but the N-terminal methionine in each tag differs from the other tag by three Daltons.
• the alpha amino group of the dipeptide tag has been guanidfnated to differentiate the fragmentation product of this amino acid from the fragmentation product of the second methionine residue and to promote protonation at this position in the tag during ionisation in a mass spectrometer.
• a further embodiment of the second aspect of this invention comprises the steps of:
• the protein sample may be digested with the sequence specific endoprotease before or after reaction of the sample with the affinity ligand mass tag.
• Phosphorylation is a ubiquitous reversible post-translational modification that appears in the majority of signalling pathways of almost all organisms as phosphorylation is widely used as a transient signal to mediate changes in the state of individual proteins. It is an important area of research and tools which allow the analysis of the dynamics of phosphorylation are essential to a full understanding of how cells responds to stimuli, which includes the responses of cells to drugs.
• Dithiol linkers have also been used to introduce fluorescein and biotin into phosphoserine and phosphothreonine containing peptides (Fadden P, Haystead TA, Anal Biochem 225(1): 81-8, "Quantitative and selective fluorophore labelling of phosphoserine on peptides and proteins: characterization at the attomole level by capillary elecfrophoresis and laser-induced fluorescence.” 1995; Yoshida O. et al., Nature Biotech 19: 379 - 382, "Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome", 2001).
• the method of Yoshida et al. for affinity enrichment of proteins phosphorylated at serine and threonine could be improved by using the maleimide tag shown in figure 8 to allow the comparison of multiple samples. This would be particularly useful for the analysis of the dynamics of phosphorylation cascades.
• a tag peptide of the form shown in figure 7 would allow direct labelling of beta- eliminated phosphothreonine and phosphoserine residues without a dithiol linker.
• the tag tetrapeptide of figure 7 is derived from methionine. Different isotopically substituted forms of methionine would be used to prepare the two different tags. The total mass of each of the two tags is the same but the N-terminal methionine in each tag differs from the other tag by three Daltons.
• the alpha amino group of the dipeptide tag has been guanidinated to differentiate the fragmentation product of this amino acid from the fragmentation product of the second methionine residue and natural methionine residues in proteins and to promote protonation at this position in the tag during ionisation in a mass specfrometer.
• the tag peptide is guanidinated at the N-Terminus to provide enhanced sensitivity and to distinguish the N-terminal residue from the C-terminal residue.
• the cysteine residue provides a free thiol, which can nucleophilically attack dehydroalanine and methyldehydroalanine.
• peptides phosphorylated at serine and threonine may be analysed in a method comprising the steps of:
• the protein sample may be digested with the sequence specific endoprotease before or after reaction of the sample with the affinity ligand mass tag.
• phosphotyrosine binding antibodies can be used in the context of this invention to isolate peptides from proteins containing phosphotyrosine residues.
• the tyrosine- phosphorylated proteins in a complex mixture may be isolated using anti-phosphotyrosine antibody affinity columns.
• a protocol for the analysis of a sample of proteins, which contains proteins phosphorylated at tyrosine comprises the steps of:
• Immobilised Metal Affinity Chromatography represents a further technique for the isolation of phosphoproteins and phosphopeptides.
• Phosphates adhere to resins comprising trivalent metal ions particularly to Gallium(i ⁇ ) ions (Posewitch, M.C. and Tempst, P., Anal. Chem., 71: 2883-2892, "Immobilized Gallium (III) Affinity Chromatography of Phosphopeptides", 1999).
• This technique is advantageous as it can isolate both serine/threonine phosphorylated and tyrosine phosphorylated peptides and proteins simultaneously.
• IMAC can therefore also be used in the context of this invention for the analysis of samples of phosphorylated proteins.
• a protocol for the analysis of a sample of proteins, which contains phosphorylated proteins comprises the steps of:
• N-hydroxysuccinimide activated tag could be used to label the free alpha-amino groups.
• a sample of phosphorylated proteins may be analysed by isolating phosphorylated proteins followed by analysis of the N or C terminal peptides of the phosphoproteins.
• Techniques for the isolation of terminal peptides are disclosed in a number of patent applications, e.g. WO98/32876, WO 00/20870 and EP 01304975.4.
• a protocol for the analysis of a sample of proteins, which contains phosphorylated proteins, would comprise the steps of:
• a pair of peptides were synthesised using conventional automated synthesis techniques to illustrate the features of this invention (both starting from commercially available Fmoc- Gly-Trt-PS resin from Rapp Polymere, Germany).
• the two peptides A and B are shown in Figure 10 and will be referred to as the two Met-Met-Gly (D3) peptides.
• Deuterated methionine (Metd 3 ) is available from ISOTEC Lie, Miamisburg, Ohio, USA.
• the Fmoc reagent for use in a peptide synthesiser must, however, be synthesised manually from the unprotected deuterated methionine.
• DIPEA Diisopropylethylamine
• a light yellow powder of peptide sequence (B) resulted.
• Figure 13 shows the proposed fragmentation reaction mechanism for the products of collision induced dissociation of the model peptides A and B shown in figure 10.
• Figure 12 shows a pair of ESI MS/MS specfra generated by an LCQ ion trap mass specfrometer from Finnigan MAT. The ESI MS/MS spectra show the fragmentation products of peptides A and B. The desired b2-fragment ion (see Figure 10) has a high intensity for both substances (273 after loss of ammonia for A and 270 after loss of ammonia for B).
• Figure 14 shows and ESI-MS/MS spectrum of the fragmentation products from the analysis of a mixture of peptides A and B. A and B were present in the mixture in a ratio of 70:30 respectively.
• the viscous liquid obtained (30g) was dissolved in 100ml dichloromethane with 8,6g (75mmol) N-Hydroxysuccinimide. 15,5g (75mmol) dicyclohexylcarbodiimide (DCC) was added in portions to the reaction mixture with stirring at RT. After 17 hours, the urea was removed by filtration. The solution was washed with a 10% citric acid solution and after removing the solvent, the product was purified by chromatography (silica gel, solvent: dichloromethane/ethylacetate). The product was then crystallized from diisopropylether. Yield: 6,0g (19%). Rf: 0,77 (dichloromethane/ethylacetate : 3/1). Fp: 108-109°C.
• the reagents are peptide tags according to this invention comprising one 'tag' amino acid linked to a sensitisation group ([1] , [2], [3]), which is a guanidino-functionality, one 'mass normalisation' amino acid and in the second pair of tags, a cleavage enhancement group, which is proline in this case ([4]).
• These tags are designed so that on analysis by collision-induced dissociation (CID), the tag fragment is released to give rise to an ion with a specific mass-to-charge ratio.
• the current accepted model of peptide scission during CID requires protonation of the peptide backbone followed by nucleophilic attack of the carbonyl moiety of the protonated amide by the next N-terminal carbonyl residue in the peptide chain to form a relatively stable oxazolone leading to scission of the amide bond ([5]).
• the sensitisation enhancer is linked to the N-terminal methionine residue by an amide bond but cleavage does not take place at this amide as there is no amide correctly positioned to allow cyclisation and cleavage at this position so cleavage can only take place between the two methionine residues.
• each pair of tags allows a pair of peptides to be distinguished by MS/MS analysis.
• Each tag can also bear a reactive functionality.
• the reactive functionality, R is not specified but could be an N-hydroxysuccinimide ester, which allows for the specific labelling of amino-groups.
• R is not specified but could be an N-hydroxysuccinimide ester, which allows for the specific labelling of amino-groups.
• this reactive functionality can be easily varied to allow different biological nucleophiles to be labelled.
• the tag design can be readily modified to accommodate an affinity ligand such as biotin.
• more than two tags can be generated allowing for comparison of additional samples or for the introduction of labelled standards.
• peptides listed in Table 7, have been synthesised as if they have been completely labelled on the alpha amino group with the above tags, i.e. the tag was 'pre-incorporated' during the synthesis to test the performance of the tags independently of the labelling reactions, so that in the following examples the 'R' group shown in Figure 18a and 18b is the peptide sequence to which the tag is attached.
• the tagged peptides were analysed by ESI-MS/MS and LC-ESI-MS/MS.
• FIGs 18a and 18b show the structures of two versions of the TMT markers.
• the tags are modular comprising different functional components that correspond to individual synthetic components in the automated synthesis of these reagents.
• Each tag comprises a sensitisation group and a mass differentiated group that together comprise the 'tag fragment' that is actually detected.
• the tag fragment is linked to a mass normalisation group that ensures that each tag in a pair of tags share the same overall mass and atomic composition.
• the first and second generation tags are distinguished by the presence of an additional fragmentation enhancing group, proline, in the second generation tag.
• the tags will additionally comprise a reactive functionality (R) to enable the tag to be coupled to any peptide but in the present experiments, R is one of a number of peptide sequences.
• R reactive functionality
• the peptides shown in Table 7 were synthesised using conventional automated Fmoc synthesis techniques (both starting from commercially available Fmoc-Gly-Trt-PS resin from Rapp Polymere, Germany).
• Deuterated methionine (Metd ⁇ ) is available from lSOTEC inc, Miamisburg, Ohio, USA.
• An Fmoc-Metd- 5 reagent for use in a peptide synthesiser was synthesised manually from the unprotected deuterated methionine as described above.
• the guanidino 'sensitisation' enhancement group was synthesized as an N-hydroxysuccinimide ester (NHS-ester) as described above and added to deprotected alpha-amino groups of synthetic peptides by conventional methods during automated peptide synthesis.
• NHS-ester N-hydroxysuccinimide ester
• Table 7 Abundance ratio experiments were performed with the peptides listed above. HPLC experiments were performed with the first three peptide sequences listed above. Pairs of synthetic peptides were prepared with either the first or second TMT pre- incorporated into the peptide sequence at the ⁇ -terminus. Sequences, mono-isotopic molecular mass and mass-to-charge ratios of predominant ion species are listed for each tag.
• CID is more selective in the LCQ
• it is unfortunately limited in its use with TMTs as it is not possible to detect small CID fragmentation products of larger precursors with this type of insfrument.
• the QTOF instrument at the higher energies of collision, consecutive fragmentations were problematic.
• the series of b- or y-ion fragments that provide sequence information are further fragmented to give smaller species so that no sequence information could be obtained from the peptide.
• the first generation TMT units can only be reliably used for the purposes of quantification without peptide identification in the QTOF. This will also be true of other serial MS/MS instruments.
• Figure 19a and 19b show typical CID specfra for a peptide labelled with the first generation TMT at collision energies of 40N (Figure 19a) and 70N ( Figure 19b).
• hi 19a weak peaks in both of the 270/273 and 287/290 regions can be seen at 40N, but they do not accurately represent the abundances of the tagged peptides. Some sequence specific y-series ions can be observed though at this accelerating potential.
• the peaks corresponding to the tag fragment can be seen clearly at m/z 270 and 273 for the first generation TMT at a collision energy of 70N. At this collision energy the intensities of these peaks accurately represent the relative abundances of each peptide (see inset for zoom of the tag region in Figure 19b) but no sequence data can be determined.
• GLGEH ⁇ IDVLEG ⁇ EQF ⁇ AAK and Guanidinocaproyl-Met(D3)-Pro-Met- GLGEHNTONLEGNEQFTNAAK had ions corresponding to the [M+3H] 3+ species at mass-to-charge ratios of approximately 897 and 929 for the first and second generation tags respectively.
• the peptides were first mixed and then analysed in a QTOF instrument with the quadrupole set to alternately select ions with m/z around 897 or 929. Each selected ion was subjected to CID at increasing collision energies.
• the identification of the peptide via its b and y series can also be performed at these lower collision energies.
• Figure 20a 20b and 20c show MS and MS/MS specfra for triply charged ions of the peptide 2 (see Table 7) labelled with the first and second generation TMTs.
• the peptides were analysed in a QTOF II insfrument.
• Figure 20a shows the MS-mode TOF spectrum of the peptide mixture.
• the first quadrupole was set to alternately select ions with m/z around 897 or 929.
• the CID spectrum at 35N for Guanidfnocaproyl- Met(D3)-Met-GLGEH ⁇ IDNLEG ⁇ EQFr ⁇ AAK is shown in Figure 20b and the CID spectrum at 35N of Guanidinocaproyl-Met(D3)-Pro-Met-
• FIG 21 shows a typical CID spectrum of a peptide labelled with these tags.
• the tag fragments revealing the abundance ratios are easily seen at the expected m/z values of 287 and 290.
• CID was performed at a relatively low collision energy of 40N.
• the peaks at m/z 287 and 290 for the second generation TMT at 40N represent the relative abundances of each peptide (see inset with zoom of the relevant region of the mass spectrum).
• FIG 22 clearly shows that the charge state of the TMT tagged peptide does not affect the appearance of the tag fragments in the CID spectra of the labelled peptides.
• a peptide labelled with a first generation TMT is shown but the same result is found for the second generation tags.
• This is advantageous as it means that scanning of the spectrum can take place without complex adjustments of the scanning software to compensate for the charge state of each peptide.
• the charge state alters the mass difference between each tagged ion pair, such that for doubly charged ions the mass difference is halved, for triply charged ions the mass difference is a third of that for the singly charged ions, etc.
• Figure 23 shows data for expected and observed ratios of peptides from ESI-MS/MS analyses of the 4 peptides listed in Table 7. Peptides with both first and second generation TMTs incorporated into them were analysed. Abundance ratios were determined by analysing the peak maxima at the d3 (A) and dO (B) of the tag fragment ion peaks after peak normalization at 290 and 287 for TMT2. Measurements were made in a QTOF instrument. The table inset to Figure 23 shows expected and observed ratios the b-ion fragments from the MS/MS analysis of eluting TMT labelled peptides. It can be seen that both generations of TMT provide accurate representation of abundance ratios of the peptides in the mixtures and that the tags show linear behaviour over the entire range of peptide ratios tested.
• Figure 24 shows the co-elution of each peptide pair, peptides A and B for each peptide from Table 7, clearly seen in the C18-reverse phase HPLC traces.
• the ion currents at m/z 287 and 290 are recorded corresponding to the tag fragments from each of the TMTs.
• the bottom trace for each peptide is the total ion current.
• the elution profiles of 3 peptides monitored at each of the mass-to-charge ratios of the b 2 ions from the tag fragments are shown. It can be clearly seen that the peptide pairs elute as a single fraction. In MS/MS mode, monitoring of the tag fragment ions produces virtually identical results in each case. For each peptide pair the observed ratios matched the expected ratios to a reasonable degree.
• the ratios of the peptide pairs are conserved throughout the elution profile, which means that it is not necessary to integrate the total ion current for the eluting ions to determine the relative abundance of each peptide pair.
• the TMT technology must be at least as sensitive as other isotope labelling methods and must have a broadly similar dynamic range.
• the properties of the tags must be consistent over the whole expected dynamic range of the samples to be analysed.
• the ability of these tags to overcome noise in the mass specfrometer needed to be demonstrated.
• the intrinsic sensitivity seems to be instrument specific based on comparisons between the LCQ and QTOF in the analysis of small peptides (the tag fragments from large peptides labelled with TMTs cannot be detected on the LCQ because of the intrinsic limitations on CID with this type of instrument).
• the sensitivity with which it is possible to determine the sequence of tagged peptides does not seem to be have been significantly changed in any of the peptides tested so far. Meaningful differences in the ratios of the peptides can be detected over the entire range of concentrations tested (Figure 25).
• Figures 26a 26b and 26c show the results of a spiking experiment in which peptides pairs 3 A and 3B (500 fmol in total, in a ratio of 40:60 respectively) bearing a second generation TMT was mixed with a tryptic digest of Bovine Serum Albumin (2 pmol).
• Figure 26a shows the base peak chromatogram from analysis in the MS-mode. During the run, the first five most intensive ions analysed in MS mode were automatically fragmented in the MS/MS mode at 3 ON. The TMT peptides pairs were investigated and located on the base peak chromatogram.
• the ratio of the TMT2 fragments was then calculated from the MS/MS spectrum for the mass [M+3H] 3+ (a zoom of the tag fragments is shown in figure 9b and the whole specfrum shown in figure 9c) by comparing the intensity of the dO and d3 TMT fragment mass-to-charge ratios (287 and 290).
• the ratio of the peptides 3A and 3B was found to be 39.3% to 60.7% respectively, by comparison of the peak intensities at the fragment ion mass-to-charge ratios of 290 (d3 TMT unit) and 287 (dO TMT unit).
• the expected ratio was 40% 3A to 60% 3B, thus the peptide ratio was detected with a 1.7% error.
• the quality of the MS/MS spectrum obtained ( Figure 26b and 26c) at the low collision energy used, allows a clear identification of the peptide sequence by database searching. This experiment clearly shows that a complex mixture of tryptic peptides does not hinder the analysis of peptide pairs labelled with the 2 nd generation TMT tags and the TMTs can help to overcome noise in the sample. In addition there do not seem to be any suppression problems - ratios of peptides present in low concentrations can still be determined in the presence of other peptides that are in high concentrations.

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